Cytokines, such as IFNγ, can contribute to either protective or deleterious outcomes in the CNS, depending on the nature of the injury or antigenic trigger. For example, in many mouse models of neurotropic viral infection, including those caused by measles virus, Sindbis virus, vesicular stomatitis virus, Theiler’s murine encephalomyelitis virus, and West Nile virus, IFNγ is critical for viral clearance and recovery. In contrast, in cerebral malaria caused by the parasite Plasmodium falciparum
, IFNγ and other Th1 cytokines have been implicated in the promotion of immunopathology and exacerbation of disease (reviewed in Hunt and Grau, 2003
). Moreover, in non-pathogen associated CNS diseases such as experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis, IFNγ is considered the key causative factor in the hallmark demyelination (reviewed in Popko et al., 1997
). Surely some of the reasons for this differential impact of IFNγ within the CNS include the location, duration and amount of IFNγ produced: in viral infections, for example, production of IFNγ by infiltrating NK and T cells may be brief and focused on a relatively low number of infected cells, whereas in chronic neuroinflammatory diseases such as EAE, unremitting IFNγ production directed at a more ubiquitous antigen (such as an autoantigen) may elicit neurotoxicity. Indeed, IFNγ is known to be directly cytotoxic: gene expression changes consequent to IFNγ exposure can lead to apoptosis (reviewed in Schroder et al., 2004
). Moreover, mice that genetically cannot downregulate IFNγ responses die within two to three weeks of birth (Alexander et al., 1999
The CNS has long been considered immune privileged (owing to the relative lack of immune surveillance within the parenchyma), which may serve to protect CNS neurons, a generally non-renewable and therefore vulnerable population. However, it is increasingly appreciated that immune responses do occur in the brain. While advances have been made in the understanding of the way in which IFNγ mediates the clearance of certain neurotropic infections (e.g. Yang et al., 2006
), how neurons respond to immune mediators, and what cellular factors may affect the outcome of these cytokine interactions, warrants further study. STAT1 phosphorylation in response to IFNγ treatment has been previously evaluated in neurons (Chesler et al., 2004
; Goody et al., 2007
; Jiao et al., 2003
; Jin et al., 2004
; Kaur et al., 2003
; Kaur et al., 2005
; Wang and Campbell, 2005
). However, the potential cell-specific responses to exogenous cytokines - specifically the significance of the timing and intensity of STAT activation -has not yet been explored in unmanipulated primary neurons.
To characterize the neuronal response to exogenous IFNγ stimulation, we compared primary hippocampal neuron cultures with MEF at three levels: basal expression of IFNγ receptor subunits; bioavailability and phosphorylation of the key IFNγ signal transducers, STAT1 and STAT3; and gene expression changes in response to IFNγ exposure. We performed standard timecourse assays under conditions of continuous IFNγ exposure and following a brief pulse. In primary neurons treated with IFNγ, as opposed to identically-treated control MEF, we observed i) reduced constitutive levels of IFNγR1 receptor subunit expression and STAT1 expression; ii) delayed and muted STAT1 phosphorylation kinetics following IFNγ exposure; iii) absence of STAT3 expression and phosphorylation; iv) decreased transcriptional response of representative IFNγ-responsive genes; and v) sustained STAT1 phosphorylation and expression of representative IFNγ-responsive genes following a pulse of IFNγ. A number of these observations warrant further discussion.
In our detailed timecourse analysis of IFNγ treated neurons (), we noted the absence of the classic cyclic pattern of phosphorylated STAT1 typically observed in IFNγ treated cells. A recent study observed a biphasic response in phosphorylated STAT3 intensity in wild-type macrophages following IL-6 exposure, which was absent in macrophages lacking SOCS-3 (Wormald et al., 2006
). The investigators proposed that this lack of SOCS-3 resulted in an inability of the macrophages to suppress STAT3 phosphorylation, causing sustained activation of STAT3. We therefore speculated that similar perturbations in the negative feedback mechanisms of the neuronal IFNγ signaling pathway might naturally exist, accounting for the apparent lack of negative regulation in our timecourse experiments. Since numerous independent negative feedback pathways act to inhibit the IFNγ response, we took a functional approach by comparing the duration of STAT1 phosphorylation and changes in gene expression in neurons following a brief (30 min) pulse of IFNγ. The observations that i) STAT1 phosphorylation in pulsed neurons gradually increased over 48 h, while being rapidly attenuated in MEF; and ii) the mRNAs encoding CXCL10, IRF-1, and SOCS-1 also accumulated during this time period, further substantiated that fundamental differences in signaling and negative feedback have a direct effect on gene expression.
The extended phosphorylation of STAT1 seen in primary neurons following an IFNγ pulse may be the result of differences in any one of several mechanisms. As mentioned, the activity of negative feedback proteins, including the SOCS family, may be impaired in IFNγ-stimulated neurons, thus allowing the receptor-associated JAKs (JAK1 and JAK2) to remain active for an extended period post-stimulation. Alternatively, decreased expression of neuronal protein tyrosine phosphatases may allow the R1 subunits of the receptor complex to remain phosphorylated, thus prolonging the availability of docking sites for STAT1 activation. Finally, the rate of STAT1 inactivation via dephosphorylation (reviewed in Darnell, 1997
) in IFNγ-treated neurons may be delayed, allowing the nuclear accumulation of phosphorylated STAT1 over time. Regardless of the mechanism, it is important to note that similar responses have been observed in rat pancreatic islet cells pulsed with IFNγ (Heitmeier et al., 1999
). In these treated cells, STAT1 was still phosphorylated and localized to the nucleus 7 days post-pulse, though the reasons for this sustained response were not addressed. Nevertheless, the ability of cells to modulate the duration of response to exogenous cytokines may be an important parameter in understanding cell-specific patterns in host immunity.
In our studies, we were surprised to note a substantial difference in neuronal expression levels of the IFNγR1 subunit (). The cellular differences observed in IFNγR1 RNA do not necessarily imply differences in protein expression. In a study of cultured dorsal root ganglia neurons, Wekerle and colleagues detected robust expression of both IFNγ subunits (Neumann et al., 1997
); thus, either subunit expression is neuron-subtype dependent, or our RNA studies do not parallel protein levels. However, even if the level of the IFNγR1 subunit is lower in neurons, functional complexes can be made, since we show in that the low available levels of STAT1 in neurons can still be activated in IFNγ stimulated neurons. How lower expression of this one subunit may impact on the neuronal response to exogenous cytokines is a matter of current study.
An important technical aspect of our study is the use of primary cells. While cell lines have been invaluable for defining key steps in cytokine responsiveness, evaluating otherwise unmanipulated, pure primary cultures may be more powerful in resolving the basis of cellular heterogeneity in cytokine responses. For example, Kaur et al. found that although treatment of a human neuroblastoma cell line with IFNγ for 30 min resulted in weak phosphorylation of STAT3, STAT3 phosphorylation was undetectable in IFNγ-treated primary rat sympathetic neurons (Kaur et al., 2003
). Thus, as our studies progress, continued use of primary neurons will be essential, not only to reveal how altered signaling impacts the eventual neuronal response, but also to ascertain whether potential differences exist in distinct neuronal subpopulations.
While these data indicate that cell-specific differences in basal expression of key signaling molecules can dramatically alter the cellular response to exogenous cytokines, care must be taken not to over-interpret these findings. For example, recent studies (Hurgin et al., 2007
; Jarosinski et al., 2001
; Massa et al., 2006
) have shown that neurons are recalcitrant to NF-kB activation. As many IFNγ-responsive genes also possess promoter elements to which NF-kB can bind, the convergence of multiple signaling pathways, such as the STAT and NF-kB pathways, likely governs the individual cellular response to exogenous cytokines. While our studies suggest cell-specific differences in STAT signaling, the contribution of other signaling pathways in cytokine responsiveness must also be considered.
In summary, we have shown that cell-specific variations in IFNγ signaling pathways, including bioavailability of key signaling effectors, strongly influence gene expression. These data further aid our understanding of why potent cytokines such as IFNγ may have apparently paradoxical effects under different circumstances. For example, perhaps less rapid and robust induction of IFNγ-responsive genes, many of which can be cytotoxic, may be advantageous for CNS neurons, and may afford some degree of protection under circumstances of chronic inflammatory challenges. Obviously, these ex vivo studies require confirmation in vivo, but we speculate that altered signaling pathways may act as a buffer between exogenous cytokines and the neuronal response. These variations in signal transduction span from receptor expression to nuclear localization of transcription factors, ultimately impacting on the initiation, intensity, duration, and profile of downstream gene expression. While the data presented in this paper pertain to the role of STAT1 in type II interferon signaling, STAT1 also plays a central role in target cell response to type I interferons. We would therefore predict that the observations presented here are pertinent to the neuronal response to type I interferons as well. An appreciation of how cells respond to soluble immune mediators will be crucial for the development of immune-based therapies appropriately tailored to the antigenic stimulus.